This invention is generally in the field of methods for the treatment and prevention of inflammatory responses involving binding reactions with the selectin GMP-140.
As described in U.S. Ser. Nos. 07/554,199 and 07/320,408, the teachings of which are incorporated herein, the adherence of platelets and leukocytes to vascular surfaces is a critical component of the inflammatory response, and is part of a complex series of reactions involving the simultaneous and interrelated activation of the complement, coagulation, and immune systems.
Endothelium exposed to "rapid" activators such as thrombin and histamine becomes adhesive for neutrophils within two to ten minutes, while endothelium exposed to cytokines such as tumor necrosis factor and interleukin-1 becomes adhesive after one to six hours. The rapid endothelial-dependent leukocyte adhesion has been associated with expression of the lipid mediator platelet activating factor (PAF) on the cell surface, and presumably, the appearance of other endothelial and leukocyte surface receptors. The slower cytokine-inducible endothelial adhesion for leukocytes is mediated, at least in part, by an endothelial cell receptor, ELAM-1, that is synthesized by endothelial cells after exposure to cytokines and then transported to the cell surface, where it binds neutrophils. The isolation, characterization and cloning of ELAM-1 is reviewed by Bevilacqua, et al., in Science 243, 1160-1165 (1989). A peripheral lymph node homing receptor, also called "the murine Mel 14 antigen", "Leu 8", the "Leu 8 antigen" and "LAM-1", is another structure on neutrophils, monocytes, and lymphocytes that binds lymphocytes to high endothelial venules in peripheral lymph nodes. The characterization and cloning of this protein is reviewed by Lasky, et al., Cell 56, 1045-1055 (1989) (mouse) and Tedder, et al., J. Exp. Med. 170, 123-133 (1989).
GMP-140 (granule membrane protein 140), also known as PADGEM, is a cysteine-rich and heavily glycosylated integral membrane glycoprotein with an apparent molecular weight of 140,000 as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). GMP-140 was first purified from human platelets by McEver and Martin, J. Biol. Chem. 259: 9799-9804 (1984). The protein is present in alpha granules of resting platelets but is rapidly redistributed to the plasma membrane following platelet activation, as reported by Stenberg, et al., (1985). The presence of GMP-140 in endothelial cells and its biosynthesis by these cells was reported by McEver, et al., Blood 70(5) Suppl. 1: 355a, Abstract No. 1274 (1987). In endothelial cells, GMP-140 is found in storage granules known as the Weibel-Palade bodies. GMP-140 (called PADGEM) has also been reported to mediate the interaction of activated platelets with neutrophils and monocytes by Larsen, et al., in Cell 59, 305-312 (October 1989) and Hamburger and McEver, Blood 75: 550-554 (1990).
The cDNA-derived amino acid sequence, reported by Johnston, et al., in Cell 56, 1033-1044 (Mar. 24, 1989), and in U.S. Ser. No. 07/320,408 filed Mar. 8, 1989, indicates that it contains a number of modular domains that are likely to fold independently. Beginning at the N-terminus, these include a "lectin" domain, an "EGF" domain, nine tandem consensus repeats similar to those in complement binding proteins, a transmembrane domain (except in a soluble form that appears to result from differential splicing), and a cytoplasmic tail.
When platelets or endothelial cells are activated by mediators such as thrombin, the membranes of the storage granules fuse with the plasma membrane, the soluble contents of the granules are released to the external environment, and membrane bound GMP-140 is presented within seconds on the cell surface. The rapid redistribution of GMP-140 to the surface of platelets and endothelial cells as a result of activation suggests that this glycoprotein could play an important role at sites of inflammation or vascular disruption.
ELAM-1, the homing receptor, and GMP-140 have been termed "selecting", based on their related structure and function.
The in vivo significance of platelet-leukocyte interactions has not been studied carefully. However, in response to vascular injury, platelets are known to adhere to subendothelial surfaces, become activated, and support coagulation. Platelets and other cells may also play an important role in the recruitment of leukocytes into the wound in order to contain microbial invasion. Conversely, leukocytes may recruit platelets into tissues at sites of inflammation, as reported by Issekutz, et al., Lab. Invest. 49: 716 (1983).
The coagulation and inflammatory pathways are regulated in a coordinate fashion in response to tissue damage. For example, in addition to becoming adhesive for leukocytes, activated endothelial cells express tissue factor on the cell surface and decrease their surface expression of thrombomodulin, leading to a net facilitation of coagulation reactions on the cell surface. In some cases, a single receptor can be involved in both inflammatory and coagulation processes.
Proteins involved in the hemostatic and inflammatory pathways are of interest for diagnostic purposes and treatment of human disorders. However, there are many problems using proteins therapeutically. Proteins are usually expensive to produce in quantities sufficient for administration to a patient. Moreover, there can be a reaction against the protein after it has been administered more than once to the patient. When, as appears to be the case for both ELAM-1 and GMP-140, as first described in U.S. Ser. No. 07/320,408, the receptor bound by the protein is a glycoprotein and the carbohydrate portion of the receptor plays a major role in the binding process, it is desirable to develop carbohydrate molecules which can be used both in vitro and in vivo to modulate binding by the selecting, as effectively as the protein molecules, but which are less expensive to synthesize, more reproducible and presumably potentially less likely to cause a reaction. In U.S. Ser. No. 07/320,408, it was proposed that inflammatory responses could be modified by employing sugars or oligosaccharides in the ligand or counterreceptor for GMP-140 on leukocytes. This was based on the presence of an N-terminal domain homologous to Ca.sup.2+ -lectins in GMP-140 and a similar domain in the lymphocyte homing receptor, for which there was evidence supporting a lectin-like interaction with target cells in which sialic acid might play an important role.
U.S. Ser. No. 07/554,199 disclosed that the ligand for GMP-140 contained critical sialic acid residues. This was based on reduction of binding of .sup.125 I!GMP-140 to neutrophils pretreated with neuraminidase from Vibrio cholera. This neuraminidase cleaves sialic acid at both .alpha.2,3 and .alpha.2,6 linkages. It was proposed that the oligosaccharide structure might include the entity, sialyl Le.sup.x, which is NeuAc.alpha.2,3Gal.beta.1,4(Fuc.alpha.1,3)GlcNAc.beta.1-R. This was based on the fact that the Le.sup.x structure, Gal.beta.1,4(Fuc.alpha.1,3)GlcNAc.beta.1-R, is a common oligosaccharide structure on myeloid cells but not on lymphocytes or erythroid cells. The trisaccharide Le.sup.x is often found in repeating units, or polylactosamine chains, which are extended oligosaccharide structures on myeloid glycoproteins or glycolipids. The neuraminidase data indicated that Le.sup.x per se could not be the ligand, but it was possible that the ligand could be sialyl Le.sup.x, or a longer sialylated, polyfucosylated polylactosaminoglycan of which sialyl Le.sup.x might be a part.
In November 1990, Larsen, et al, in Cell 63: 467-474 (1990), claimed that Le.sup.x is, or is a major "component" of the ligand, based on inhibition of neutrophil or HL60 cell binding to activated platelets (which express GMP-140) or to COS cells transfected with GMP-140 cDNA. The inhibition was either with high concentrations of monoclonal antibodies to Le.sup.x or with concentrations of LNFIII up to 300 .mu.M. LNFIII is Gal.beta.1,4(Fuc.alpha.1,3)GlcNAc.beta.1,3Gal.beta.1,4Glc; thus it includes the Le.sup.x trisaccharide.
Corral, et al., in Biochem. Biophys. Res. Comm. 172(3), 1349-1356 (November 1990), also stated that sialic acid residues are a key feature of the carbohydrate ligand for GMP-140. Their assay was the rosetting of activated platelets to neutrophils or HL60 cells. They could inhibit rosetting by pretreating neutrophils with neuraminidase from Vibrio cholera, in agreement with the findings disclosed in U.S. Ser. No. 07/554,199. However, they did not prevent rosetting with neuraminidase from Newcastle disease virus. Thus they inferred that the linkage is likely to be .alpha.2,6. However, since the Newcastle enzyme is part of an intact virus it is probable that the virus directly agglutinated platelets to neutrophils, obscuring the inhibitory effect of the neuraminidase.
At least four papers have appeared on the carbohydrate ligand for the related selectin, ELAM-1: Phillips, et al., Science 250: 1130-1131 (November 1990); Walz, et al., Science 250: 1132-1135 (November 1990); Lowe, et al., Cell 63: 475-484 (November 1990); and Goelz, et al., Cell 63: 1349-1356 (December 1990). These reports conclude that the ELAM-1 ligand is either sialyl Le.sup.x or a related sialylated fucosylated structure. All of the reports describing the ligand for ELAM-1 agree that it is a sialylated fucosylated structure, with at least some of the reports suggesting that the ligand is sialyl Le.sup.x per se.
Brandley, et al., in Cell 63: 861-863 (November 1990) review what is known about the ligands for the various selecting and the potential applications for these structures.
It is therefore an object of the present invention to provide the carbohydrate structures forming a part of the ligand or counterreceptor for the selectin GMP-140, which is distinct from the other selectins such as ELAM-1.
It is another object of the present invention to provide methods for using these carbohydrate structures to inhibit leukocyte adhesion to endothelium or to platelets.
It is a further object of the present invention to provide methods for using these carbohydrate structures to modulate the immune response and the hemostatic pathway.